1. Field of the Invention
The present invention relates to a method and apparatus for separating red blood cells from whole blood.
The present application is related to application Ser. No. 08/049,862, filed Apr. 20, 1993, the entire contents of which are hereby incorporated by reference.
2. Description of the Background Art
The ability to measure a wide variety of physiologically active compounds, both naturally occurring and synthetic, has become of increasing importance, as an adjunct to both diagnosis and therapy. While for the most part such assays have required clinical laboratory determinations, there is an increasing awareness of the importance of being able to conduct assay determinations in a physician's office or in the home. These environments require that the assay have a simple protocol and be relatively free of sensitivity to small changes in the conditions under which the assay is carried out. Importantly, inaccurate measurements of reagents and sample should, whenever feasible, be avoided. A number of systems have been developed to address the various problems associated with analysis outside of the clinical laboratory.
One analyte of importance is cholesterol. There is a clearly established relationship between total blood cholesterol (mainly the LDL fraction) and coronary artery disease (J.A.M.A. 253: 2080-2086, 1985). New guidelines have been established for adults to identify risk groups associated with blood cholesterol levels. Since cholesterol levels can be controlled by both diet and cholesterol lowering drugs, for those individuals at risk, the ability to monitor one's own cholesterol at home is useful in reducing the potential for heart disease. The measurement of other naturally occurring compounds of physiological importance, such as glucose, lipoproteins, etc., as well as synthetic drugs, is also of great interest, as assays become more sensitive and control can be exercised more diligently by the patient.
In clinical assays, the separation of serum or plasma from whole blood is extremely important, since it is often difficult to conduct the analysis of dissolved blood components without interference from the red blood cells. Serum or plasma is conventionally separated from erythrocytes by centrifuging. Centrifugation, however, causes other problems because one must then separate the supernatant from the blood cake. Moreover, this method is not available for use in home or office diagnostic assays.
Using whole blood with diagnostic devices for use in home or office assays gives rise to further problems. In these devices, it is customary to employ reagents which cause a color change if the analyte is present (or, alternatively, if it is absent). Turbid or colored solution, such as whole blood, may interfere with the readings.
Means for the fractionation of whole blood into blood cell plasma fractions are known in the art.
Vogel et al., in U.S. Pat. No. 4,465,24, disclose a process for separating plasma or serum from whole blood using a filter made of glass fibers. The glass fibers used have an average diameter of 0.2 to 5 microns, and a density of about 0.1 to 0.5 g/cm. Whole blood is placed onto a layer of glass fibers, and plasma is generated by retardation of flow of the cells. Plasma is collected at the other side of the glass fibers.
Another approach to separating red blood cells from whole blood is shown in Hillman et al., U.S. Pat. No. 4,753,776. In this patent, capillary action is used to pull whole blood through a glass microfiber filter by retarding the flow of the cells.
Allen et al., in U.S. Pat. No. 4,987,085, disclose a device and method for separating plasma from whole blood via a filtering system with descending pore size to provide for successive removal of red blood cells without lysis. A combination of glass fiber membranes and cellutosic membranes is used to minimize red blood cell lysis while removing red blood cells.
Kondo et al., in U.S. Pat. No. 4,256,693, disclose a multilayered integral chemical analysis element for blood comprising a filter layer capable of removing formed components from the blood. The filter layer may be made of at least one component selected from paper, nonwoven fabric, sheet-like filter material composed of powders or fibers such as man-made fibers or glass fibers, and membrane filters having suitable pore sizes. The filter layer separates the formed components of the blood at one time, or successively, such as in the order of leukocytes, erythrocytes, and platelets.
Other filtration systems are described in U.S. Pat. Nos. 3,092,465; 3,630,957; 3,663,374; 4,246,693; 4,246,107; and 2,330,410. Some of these filters employ membranes with small pores. A disadvantage of these filters is that blood can only penetrate through the membrane filter very slowly and in small amounts, because the membrane is very easily blocked. This results in a reaction which takes longer than is desirable.
Unfortunately, blood separation devices using glass fiber filters or membranes tend to retain significant amounts of serum or plasma, and display a relatively slow speed of separation. This presents a problem with diagnostic devices which are quantitative, as there must be sufficient sample present in the detection area to provide a reliable indication of the quantity of analyte detected. If insufficient sample flows through the filter, a false low reading will be obtained. Moreover, devices intended for home or office use should be convenient to use and should provide an indication of the analyte within a reasonably short period of time. It is thus essential to remove unwanted red blood cells to allow most of the remainder of the blood to pass through the separation device, and then filter the blood relatively quickly.
To solve these problems, test papers have been coated with semi-permeable membranes (U.S. Pat. No. 3,092,465), and swellable films into which only the dissolved components of the blood can penetrate, leaving the erythrocytes (U.S. Pat. No. 3,630,957). These two methods are only useful for testing low molecular weight components of blood such as glucose or for urea. Higher molecular weight components of the blood such as lipids, or substrates bound to serum protein, such as bilirubin, cannot be determined in this way because they are not able to penetrate into the film or to pass through the semipermeable membrane.
Alternative solutions include covering diagnostic agents with membrane filters for separating the blood cells, as disclosed in U.S. Pat. Nos. 3,663,374 and 4,246,693. A disadvantage with these diagnostic agents is that the blood can only penetrate through the membrane filter very slowly and in small amounts, because the membrane is very easily blocked. This results in a reaction which takes longer than is desirable.
U.S. Pat. Nos. 4,246,107 and 2,330,410, teach that lymphocytes and leukocytes can be separated from blood when the blood is filtered through a layer of synthetic resin fibers with an average fiber diameter of from 5 to 20 microns for lymphocytes, and from 3 to 10 microns for leukocytes. However, since the erythrocytes preponderantly pass through the filter with the plasma, these filters are not suitable for obtaining plasma.
Red blood cells can also be removed from whole blood samples by contacting a whole blood sample with an agglutinating agent. One type of red blood cell agglutinating agent is a lectin, a family of sugar binding protein first identified in plants. Red blood cells can be removed from whole blood by contacting the whole blood with a lectin, which attaches itself to specific glycoproteins on the red blood cell membrane and forms large masses by agglutination of the cells. However, when lectins contact red blood cells, cross-linking occurs to form a gel. The presence of this gel in a filter greatly reduces the flow of blood through a filter, and therefore limits the amount of plasma recovered.
Sand et al., in U.S. Pat. No. 5,118,428, disclose using an acid, such as citric, acetic or ascorbic acid, to agglutinate the red blood cells for separation from whole blood. However, use of an acid lowers the pH of the plasma, which may interfere with subsequent analyses.
Trasch et al., in U.S. Pat. No. 5,055,195, disclose the use of erythrocyte-retention substrates which contain two strongly polar groups which are connected by a non-polar bridge which serves as a spacer. These substrates change the polarity of the surface of the erythrocytes and cause them to agglutinate. The agglomerates formed in the blood can then be readily separated by filtration.
Zuk, in U.S. Pat. No. 4,594,327, teaches a method for separating red blood cells from whole blood by combining the whole blood sample with a red-blood cell binding agent. This mixture is then filtered through a solid bibulous agent.
Rapkin et al., U.S. Pat. No. 4,678,757, disclose a method for separating blood into fluid and cellular fraction for diagnostic tests. The whole blood is introduced into a carrier containing a layer of carbohydrate, which separates the fluid from the cellular fractions.
Hillman et al., in U.S. Pat. Nos. 4,753,776 and 5,135,719, disclose a method for separating plasma from red blood cells wherein a low pressure filter is interposed in a pathway between an inlet port and a reaction area. Capillary force is the sole driving force for the movement of plasma from the filter to the reaction area. The filter is made of glass microfiber filters which can operate in the presence or absence or agglutins.
A device for separating plasma or serum from whole blood is disclosed in Aunte et al., U.S. Pat. No. 4,933,297. This device consists of a matrix of hydrophilic sintered porous material with at least one red blood cell agglutinating agent incorporated therein. An optional filter containing the same agglutinating agent is added to give a filter combination that yields plasma which is about 97% free of red blood cells.
Laugharn et al., in U.S. Pat. No. 4,946,603, describe a filter means to retain blood cells which pass through a matrix of hydrophilic sintered porous material to which at least one red blood cell agglutinating agent has been applied. The agglutinating agents used include natural and synthetic water soluble polymers, including hexadimethriine bromide, polylysine, and anti-red blood cell antibodies. The agglutination process is enhanced by incorporating substances such as polyvinyl pyrrolidone which functions as a dielectric, allowing charged cells to approach one another and by crosslinking by antibody and/or other agglutinins.
Many of the most commonly used assays in disposable assay devices require an incubation step, such as requiring enzymes to act on the sample, such as determinations of cholesterol, glucose, uric acid, and the like. Additionally, enzymes are often used as labels in immunoassay. In a conventional enzyme immunoassay, an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal generated by the enzyme, in either the conventional or the immunoassay, may be a color change, detected with the naked eye or by a spectrophotometric technique.
However, despite many attempts by prior workers to provide means to separate red blood cells from whole blood quickly and efficiently, there are still many disadvantages to the methods described above.
Ideally, a disposable assay device should include a means to delay the flow of the sample through the device for a predetermined time to permit incubation of the sample with the reagents or indicators present in a particular region of the device. After the incubation period, which is generally on the order of a few minutes or less, the sample then flows to the next region of the device for further processing. An ideal flow delay means should work like a valve, with a "closed" and an "open" state. When the state is "closed", the fluid flow should stop, and when the state switches to "open", the fluid should flow through the flow-delay valve with little or no restriction, and the flow rate of the fluid through the device should be substantially unchanged.
A wide range of disposable analytical devices have been developed which include means to control flow of fluids therethrough. However, none of these previously developed devices has a valve-like means to control the flow of fluids.
Although there has been no previous disclosure of assay devices including a valve-like means, a number of disposable analytical devices include means for delaying flow. Examples of these can be found in Deutsch et al., U.S. Pat. No. 4,522,923; Ebersone, U.S. Pat. No. 4,522,786; Jones, U.S. Pat. No. 5,213,965; Vonk, U.S. Pat. No. 5,185,127.
Assay devices in which contact between layers is delayed by physically separating the layers until the operator places the layers into contact with each other are shown in Deneke et al., U.S. Pat. No. 4,876,076; and Ramel et al., U.S. Pat. No. 4,959,324.
Other means for controlling flow of samples through assay devices include those of Woodrum, U.S. Pat. No. 4,959,305, reversibly immobilized assay reagents; Columbus, U.S. Pat. No. 4,549,952, viscosity increasing means; Bruce et al., Analytical Chemica Acta 249 (1991), 263.269, expansion of compressed foam; Hillman et al., U.S. Pat. No. 4,963,498, agglutination of binding pairs; Kurn et al., U.S. Pat. No. 5,104,812, interrupting capillary flow.
Physical barriers to interrupt flow are shown in Columbus, U.S. Pat. Nos. 4,310,399 and 4,618,476 and Grenner et al., U.S. Pat. No. 5,051,237.
Liquid flow through a filter is controlled by reactions between the sample and a component of the filter, such as in Marchand et al., U.S. Pat. No. 5,127,905 and Tanaka et al., U.S. Pat. No. 4,966,784.
Impregnated layers which are not valve-like in their actions are shown in Liotta, U.S. Pat. No. 3,723,064; Engelmann, U.S. Pat. No. 4,738,823; Nagatomo et al., U.S. Pat. No. 4,587,102; Koyama et al., U.S. Pat. No. 4,615,983; Rothe et al., U.S. Pat. No. 4,587,099; Nelson, U.S. Pat. No. 4,923,680.
Moreover, none of the above-noted patents provides a reliable means for metering the rate of flow delay through a layer in order to retain a sample in contact with a reagent for a predetermined length of time.
No admission is made that any of the patents or other background art cited above constitutes prior art.